Effects Of Achaete-scute Homolog 1(Ascl1) On The Proliferation And Invasion In Human Gioma Cells

Objective: To investigate the effect of Achaete-scute homolog 1(Ascl1)on the proliferation and invasion in human gioma cells(U87 cells),and to explore the regulation mechanism.Methods: The Ascl1 gene was integrated into the U87 cell chromosome using lentiviral vector to construct stable Ascl1 overexpression U87 cell line.The overexpression results of Ascl1 in U87 cells were identified by observing the expression of green fluorescence,Western Blot and Q-PCR.CCK-8 and BrdU assays were used to determine the effect of Ascl1 on the proliferation in U87 cells.The effect of Ascl1 on the invasion in U87 cells was checked by transwell invasion assay.The cell cycle of U87 cells regulated by Ascl1 was investigated by flow cytometry,and Western blot was taken to detect the expression of c-myc,Cyclin D1,CDK4 and P27 in each group,which could preliminarily revealed the mechanism of Ascl1 on the proliferation and invasion in U87 cells.Results: Through observing the green fluorescence expression,Western Blot and Q-PCR experiments,it was determined that Ascl1 was overexpressed in U87 cells after transfection of lentivirus.The transfection efficiency exceeded 90 percent.Ascl1 gene was successfully integrated into the U87 cell chromosome,and the Ascl1 overexpression U87 cell line was constructed.Compared with the control group cells,CCK-8 results showed that the optical density(OD)at 450 nm decreased in Ascl1 overexpression U87 cells(P < 0.01),the results from BrdU assay demonstrated that BrdU-positive cell rate also decreased in Ascl1 overexpression U87 cells(P < 0.01),suggesting that Ascl1 inhibited the proliferation of U87 cells.Transwell invasion assay showed that the number of cells which passed through the membrane was reduced in Ascl1 overexpression U87 cells,compared with the control group cells(P < 0.01),suggesting that Ascl1 suppressed the invasion of U87 cells.Flow cytometry demonstrated that the cell cycle of Ascl1 overexpression U87 cells was arrested in G0 /G1 phase,and G1 phase to S phase conversion was inhibited(P < 0.01);at the same time,Ascl1 down-regulated the expression of c-myc,Cyclin D1 and CDK4,and up-regulated the expression of P27(all P <0.01),indicating that the effect of Ascl1 on the proliferation and invasion of U87 cells was related to cell cycle arrest.Conclusion: Ascl1 can reduce the proliferation and invasion in U87 cells,which is related to the regulation of cell cycle.